Recent studies show that HIV-1 Vif acts by overcoming the antiviral activity of APOBEC3G, a cytidine deaminase that induces G to A hypermutation in newly synthesized viral DNA. In the absence of Vif, APOBEC3G incorporation into virions renders HIV non-infectious. Our lab and others recently demonstrated that Vif overcomes the antiviral activity of APOBEC3G by targeting it for destruction by the ubiquitin-proteasome pathway. Preliminary studies suggest that Vif binds directly to APOBEC3G and forms a complex with Cullin 5 (Cul5), elongin B, and elongin C, suggesting that Vif induces APOBEC3G degradation through a Cul5 E3 ubiquitin ligase complex. Vif contains a conserved BC-boxlike domain found in other proteins that interact with cullin-elongin BC complexes. Deletion of the SLQYLA sequence in the BC-box-like domain of Vif disrupts its interaction with Cul5 complexes and also abrogates its ability to induce APOBEC3G degradation and enhance viral infectivity. These findings suggest that Vif functions like an F-box, serving as a bridge between APOBEC3G and a Cul5-based E3. The overall goal of this application is to understand the mechanism by which Vif induces the degradation of APOBEC3G via the ubiquitin-proteosome pathway. The specific aims are: 1) Characterize the HIV-1 Vif-Cullin 5-containing complex (Vif-Cul5 complex) and determine which cellular protein in this complex interacts directly with Vif; 2) Determine how APOBEC3G is targeted to the Vif-Cul5 complex; 3) Determine whether the ubiquitination of APOBEC3G by Vif-Cul5 complexes can be reconstituted in vitro; 4) Define the role of a novel zinc binding site in Vif in binding to elonginBC-Cul5 complexes and APOBEC3G, and Vif mediated degradation of APOBEC3G; 5) Further characterize the mechanism by which Vif induces APOBEC3G degradation. These studies will provide important insights into the mechanism by which Vif overcomes the anti-viral activity of APOBEC3G, and therefore have significant implications for identifying new therapeutic targets.